PROJECT SUMMARY/ABSTRACT The host T cell response to HBV is vigorous and multi-specific in acutely infected patients who clear the virus, but it is weak and more narrowly focused in those who become chronically infected. Therefore, although the human immune response is capable of eliminating the infection, it often fails to do so. Therapeutic immunization to induce an immune response sufficient to control the virus is a possible new approach for treating chronic hepatitis B. Unfortunately however the current HBV vaccine is not effective for therapeutic vaccination. Although it elicits a strong neutralizing antibody response that prevents infection, the current vaccine does not induce the potent CD8 T cell response needed to eliminate the virus after infection. An important step forward would be to develop immune therapy approaches that induce both CD8 cellular immunity and effective antibodies in vaccinated individuals. The VLV technological innovation under development is capable of inducing both a robust antibody responses as well as multi-specific CD8 T-cell responses addressing the need for HBV immunotherapy. We have previously found that an improved version of the reverse-engineered platform that generates ?virus-like vesicles? (VLVs) containing VSV-G but no other viral structural proteins are safe, are genetically stable and lack neurovirulence in mice. Employing this evolved VLV vector engineered to express the HBV middle surface envelope glycoprotein (MHBs) we have found that it induces CD8 T cell responses in mice that were greater in magnitude and broader in specificity than those obtained with other immunization strategies, including recombinant protein and DNA. A prime-boost immunization enhanced CD8 T cell responses in nave mice and that regimen induced HBV-specific CD8 T cells in a transgenic mouse model of CHB infection. We have rationally designed and engineered a VLV vaccine that encompasses multiple antigens of the HBV genome and show that this multimeric vaccine induces CD8- specific T cells in a single immunization. We have further engineered these polyproteins to be secreted. We will now test the hypothesis that the secreted versions of the non-secreted VLV-Multi-Antigen constructs will not only drive both a superior cell-mediated and antibody immunity but would also increase the breadth and magnitude of these responses when compared to a single antigen MHBs antigen construct. To evaluate our hypothesis, we will carry out three specific aims. First, we will characterize the immune response to the VLV-Multi-Antigen vectors in normal mice. Second, we will develop and optimize a prime- boost strategy in order to enhance the immune responses to these vectors. Finally, we will establish milestones for phase II development. The propose research is significant because an effective therapeutic vaccine that cures chronic HBV would have a substantial impact on the prevention of HBV-associated chronic liver diseases.